This laboratory is titled Translational Research, as we investigate human inherited retinal degenerations identified in the clinic as both a source of information about retinal function and dysfunction and as a target for research in therapeutic intervention. Current efforts focus on human X-linked juvenile retinoschisis (XLRS). XLRS is an inherited disease and is a leading cause of juvenile macular degeneration in human males. It is due to mutations in the retinoschisin (RS) gene found on the X chromosome. We are working to understand the disease mechanisms that bring about retinal structural changes and neuronal synaptic signaling deficiency in a mouse model created in this laboratory section. At the same time, we are carrying out gene transfer therapy using a viral vector to supply a normal copy of the retinoschisin gene to the retina of patients in which it is defective. Our current understanding is based on a study of human affected patients and by analysis of the XLRS animal model, which is a retinoschisin gene knockout (Rs1-KO) mouse. We probed the biochemistry and sub-cellular localization of the retinoschisin protein and have localized it to particular cell membrane sites of photoreceptors and synapses and measured changes in key membrane proteins in synapses. We discovered molecular interactions between retinoschisin and photoreceptor membrane phospholipids biochemically and with atomic force microscopy that may explain its role in neuronal structure and retinal signaling. We cloned and characterized the human gene promoter region and have identified the key regulatory sites. We characterized the biochemical consequences of certain human mutations in the RS gene, and showed that they lead to an absence of the protein. We have identified mutations that produce more severe and less severe clinical phenotypes. Detailed study of long-term disease progression in the XLRS mouse revealed significant correlations between degenerative structural changes and functional neuronal signaling abnormalities. Our recently published study on synaptic pathology and therapeutic repair in adult retinoschisis mouse we show that RS1 protein deficiency in XLRS causes a unique pattern of molecular failure at the connection between retinal neurons, the synapse, different from that of other mouse models of synaptic dysfunction that limit vision. We found that molecular pathology could be reversed upon provision of the RS1 protein by gene transfer to the adult XLRS mouse retina and that this restored the normal resting potential of postsynaptic neurons. Such studies currently are not possible in human and provide us better understanding of disease mechanisms and give clues on designing appropriate endpoint metrics for eventual human clinical trial. In preparation for a clinical treatment trial for XLRS by viral (AAV) vector retinoschisin gene transfer, we characterized appropriate intervention times, doses and other parameters that lead to rescue of structure and function in the XLRS mouse. We have shown that gene transfer to affected eyes leads to long term improvement of retinal structure and function as well as expression of retinoschisin protein in retinal cells. We have shown that doses of the vector which produce significant improvement of retinal structure and function are not toxic to the eyes of mice and rabbits in an externally conducted preclinical GLP (Good Laboratory Practices) safety trial. Based on these preclinical results, the FDA approved initiation of a human clinical trial. We enrolled the first subject in the phase I/IIa, prospective, three dose escalation, single-center clinical trial with AAV-RS1 in 2015. The goal is to evaluate the safety and tolerability of ocular RS1 AAV vector (AAV8-scRS/IRBPhRS) gene transfer to the retina in participants affected with X-linked juvenile retinoschisis (XLRS). Originally, nine male participants affected with XLRS were to receive ocular gene transfer, with three participants in each of three dose cohorts. Additional participants (up to 6) could be enrolled at an identified dose that is well-tolerated and potentially efficacious for a total enrollment of up to 15 participants. One eye of each participant is receiving the RS1 gene vector administered by intravitreal injection. The study will be complete once the final participant in the last study cohort has received 18 months of follow-up. Participants will continue to be followed for up to 15 years after enrollment, or per current FDA requirements, for further safety analysis. The primary outcome is the safety of ocular RS1 AAV vector as determined from assessment of retinal function, ocular structure and occurrence of adverse events and abnormal laboratory tests. Secondary outcomes include changes in visual function, electroretinogram (ERG) responses, retinal imaging with optical coherence tomography (OCT), visual field measurements and the formation of anti-AAV or anti-RS1 antibodies. Circulating T-cell levels were also analyzed. All 9 subjects of the first protocol have been dosed. Two additional participants were dosed. One at 1 e11 vector genomes (vg) per study eye; the other with 3 e11 vg per study eye. These two participants were given an immunosuppressive regimen prophylactically prior to vector administration. Ongoing lab and clinic efforts seek to improve our understanding of the basic biology of the retinoschisin molecule as well as disease mechanism, progression, genotype-phenotype correlation and effect of treatment at different ages. We have seen positive effects of treatment in the mouse model of the XLRS at advanced age, suggesting treatment of humans at an older age could improve visual function. We have explored subtle changes in retinal morphology in the XLRS mouse in vivo using OCT to explore new ways of detecting therapeutic effects in humans. We continue to explore alternate AAV vectors, alternate promoters, different delivery methods, and repeat administration into the non-study eye to further characterize and optimize gene therapy delivery into XLRS participants. In non-human primates, we are initiating studies to characterize RS1 protein distribution in normal untreated monkey eyes and in eyes receiving gene vector. In parallel, we have initiated a series of studies to better understand the intraocular distribution and levels of RS1 in normal human eyes. Current efforts also include the generation of cells that constitutively express RS1 protein. ClinicalTrials.gov Identifier: NCT00055029 Clinical and Genetic Studies of X-Linked Juvenile Retinoschisis The objectives of this registry are to understand the nature of the XLRS disease in order to develop appropriate treatments by characterizing the anatomical and functional characteristics of retinoschisis and ultimately generate a well-documented genotype-phenotype correlation map. A minimum of 100 males diagnosed with X-linked retinoschisis will undergo clinical examination and have their blood drawn for genotyping. Blood will also be drawn from available and consenting mothers of affected males. An eye examination will be performed, and blood drawn from any symptomatic available and consenting female family members. A maximum of 500 affected males and family members may be enrolled. Sites outside of NIH are participating as referral centers to accumulate the cohort. ClinicalTrials.gov Identifier: NCT02317887 Study of RS1 Ocular Gene Transfer for X-linked Retinoschisis The objective of this registry is to see if the AAV-RS1 vector is safe to use in people. Up to 100 male participants with XLRS will be screened under this protocol. Nine male participants affected with XLRS will receive ocular gene transfer, with three participants in each cohort of three dose phases. Additional participants may be enrolled at a dose identified as well-tolerated.